WO2008135561A1 - Modified-release particles based on polyelectrolytes and on an active principle and pharmaceutical formulations containing these particles - Google Patents

Abstract

The present invention relates to novel polyelectrolyte polymer particles for transporting an active principle (AP), in particular a protein and peptide active principle, and also to novel modified-release pharmaceutical formulations containing said AP microparticles. These novel AP-loaded particles release the AP over a prolonged duration of several days, or even several weeks. The invention relates, in a first aspect, to particles comprising: a) a first polyelectrolyte polymer (PE1) in the charged state, bearing side hydrophobic groups (HG), said first polyelectrolyte polymer (PE1) spontaneously forming, in water, a colloidal solution of particles at at least a value pHm of the pH between 3 and 8; b) a second polyelectrolyte polymer (PE2) of opposite polarity to that of the first polyelectrolyte polymer (PE1), said second polyelectrolyte polymer (PE2) forming, in water, a solution or a colloidal solution at at least said pHm value of the pH; c) at least one active principle (AP) combined non-covalently with the particles of the colloidal solution of the first polyelectrolyte polymer (PE1); said particles being obtained by mixing, at a pH equal to pHm, of the first polyelectrolyte polymer (PE1) in the form of a colloidal solution of particles combined with the active principle (AP) with the second polyelectrolyte polymer (PE2) in the form of a solution or a colloidal solution. The invention also relates to the process for preparing these particles, a pharmaceutical formulation comprising such particles and a process for preparing medications.

Description

 PARTICLES BASED ON POLYELECTROL YTES AND MODIFIED RELEASE ACTIVE INGREDIENTS AND PHARMACEUTICAL FORMULATIONS CONTAINING THESE PARTICLES

FIELD OF THE INVENTION

The present invention relates to novel transporters of active (s) -PA-, in particular protein (s) and peptide (s), as well as new modified-release pharmaceutical formulations containing said PA transporters. The application also relates to the applications, particularly therapeutic applications, of these pharmaceutical formulations. These active pharmaceutical formulations concern both human and veterinary therapeutics.

The abbreviation PA used throughout this specification is for at least one active ingredient.

STATE OF THE ART

In the field of the prolonged release of pharmaceutical PAs, in particular therapeutic peptides / proteins, it is very often sought to reproduce at best in the patient a plasma concentration of peptide or protein close to the value observed in the healthy subject.

This objective is hampered by the short lifespan of the proteins in the plasma which leads to the repeated injection of the therapeutic protein. The plasma concentration of therapeutic protein then has a "sawtooth" profile characterized by high concentration peaks and very low concentration minima. Concentration peaks, much higher than the basal concentration in healthy subjects, have very marked adverse effects because of the high toxicity of therapeutic proteins such as certain cytokines. In addition, the concentration minima are lower than the concentration required to have a therapeutic effect, resulting in poor therapeutic coverage of the patient and serious long-term side effects.

Also, to reproduce in the patient a plasma concentration of therapeutic protein close to the ideal value for the treatment of the patient, it is important that the pharmaceutical formulation considered allows the release of the therapeutic protein over a prolonged period, so as to limit the variations in concentration. plasma over time.

Furthermore, this active formulation should preferably satisfy the following specification, already known to those skilled in the art:

1 - sustained release of an active and undenatured therapeutic protein, by human or synthetic example, so that the plasma concentration is maintained at the therapeutic level;

2 - injection viscosity sufficiently low to be easily injectable;

3 - biocompatible and biodegradable form having an excellent toxicity and tolerance profile.

In an attempt to achieve these objectives, one of the best approaches proposed in the prior art was to develop sustained-release forms of therapeutic protein (s) consisting of suspensions, liquid and low viscosity nanoparticles loaded with therapeutic proteins. These suspensions allowed the easy administration of native therapeutic proteins.

Thus, Flamel Technologies has proposed a route in which the therapeutic protein is associated with nanoparticles of a copolyamino acid comprising hydrophobic groups and hydrophilic groups. The patent application US 2006/0099264 discloses amphiphilic polyamino acids comprising aspartic acid residues and / or glutamic residues, at least a portion of these residues carrying grafting agents comprising at least one alpha-tocopherol unit, eg: (polyglutamate or polyaspartate grafted with alpha tocopherol of synthetic or natural origin). These "hydrophobic modified" homopolyamino acids spontaneously form in water a colloidal suspension of nanoparticles, which are able to associate easily in aqueous suspension at pH 7.4, with at least one active protein (insulin).

The in vivo release time of the (or the) active protein (s) (e.g. insulin) "vectorized" by the suspensions according to US 2006/0099264 would benefit from being increased.

The increase in the release time has been partially achieved by the pharmaceutical forms disclosed in PCT application WO-A-05/051416. In this application, is disclosed a colloidal suspension of nanoparticles (0.001-0.5 microns) of hydrophobic modified poly (L-glutamate) and injected at a concentration such that after subcutaneous injection, it is formed in situ in the patient a gel in contact with endogenous albumin. The protein is then slowly released over a typical period of one week. However, when the therapeutic protein concentration to be administered is relatively high, as is the case for example for human growth hormone, the release time is limited to a few days.

The duration of liberation of these forms should be further increased. BRIEF DESCRIPTION OF THE INVENTION

One of the objectives of the invention is to propose new particles charged with AP, releasing the AP over a prolonged period of several days, or even weeks. Another object of the invention is to provide new particles forming a stable suspension in aqueous solution.

Another object of the invention is to propose new particles loaded with PA and stable in freeze-dried form.

Another object of the invention is to provide new particles capable of being preserved in freeze-dried form.

Another objective of the invention is to propose new easily redispersible particles after lyophilization.

Another object of the invention is to propose new particles releasing a protein that has preserved its biological activity. Another object of the invention is to propose a new process for the preparation of these microparticles.

Another object of the invention is to provide a solid pharmaceutical formulation for the sustained release of PA, in particular a dry powder form for inhalation and pulmonary administration. To achieve these objectives, among others, the inventors have had the merit of discovering, after long and painstaking researches, that, quite surprisingly and unexpectedly, the mixing under specific conditions of two polymers (for example copolyamino acids) polyelectrolytes of opposite polarity, at least one carrying hydrophobic groups, associated with at least one PA, leads to particles of size between 1 and 100 microns capable of releasing in vitro or in vivo a protein or a peptide over an extended period.

From which it follows that the invention relates firstly to particles for the sustained release of at least one active ingredient (AP), characterized in that they comprise: a) a first polyelectrolyte polymer (PEI) , preferably a linear alpha-polyamino acid, in a charged state, carrying hydrophobic groups (GH) on the side, said first polyelectrolyte polymer (PE1) spontaneously forming in water a colloidal solution of particles at at least a pHm value of pH inclusive between 3 and 8; b) a second polyelectrolyte polymer (PE2), preferably a linear alpha-polyamino acid, of opposite polarity to that of the first polyelectrolyte polymer (PE1), said second polyelectrolyte polymer (PE2) forming a solution or a colloidal solution in water; minus said pHm value of the pH; at the condition that, if the first electrolyte polymer (PE1) is a polyamino acid, then the second polyelectrolyte polymer (PE2) is neither polylysine nor polyethylene imine; c) at least one active ingredient (PA) non-covalently associated with the particles of the colloidal solution of the first polyelectrolyte polymer (PEI); said particles for the sustained release of at least one active ingredient (PA) being obtained by mixing, at pH equal to pHm, the first polyelectrolyte polymer (PEI) in the form of a colloidal solution of particles associated with the active principle (PA) with the second polyelectrolyte polymer (PE2) in the form of a solution or colloidal solution.

The invention also relates to a method for preparing particles for the sustained release of at least one active principle (AP), these particles being in particular those described above, comprising the following steps:

1) the preparation, at a pHm value of pH between 3 and 8, of an aqueous colloidal solution of a first polyelectrolyte polymer (PE1) in the charged state, carrying hydrophobic groups (GH) on the side, said first polymer polyelectrolyte (PE1) being capable of spontaneously forming in water a colloidal solution of particles at at least a pHm value of pH of between 3 and 8; 2) adding at least one active ingredient (AP) to the first polyelectrolyte polymer (PEI) obtained in step 1, said active ingredient non-covalently associating with the particles of the colloidal solution of said first polyelectrolyte polymer ( PEI);

3) preparing a second polyelectrolyte polymer (P E2) of opposite polarity to that of the first polyelectrolyte polymer (PE1), said second polyelectrolyte polymer (PE2) forming in water a solution or a colloidal solution at least said value pHm of the pH; with the proviso that, if the first electrolyte polymer (PE1) is a polyamino acid, then the second polyelectrolyte polymer (PE2) is neither polylysine nor polyethylene imine;

4) mixing, at pH equal to pHm, the first polyelectrolyte polymer (PEI) in the form of a colloidal solution of particles associated with the active principle (PA) obtained in step 2) with the second polyelectrolyte polymer (PE2) in the form of solution or colloidal solution obtained in step 3).

The invention also relates to particles for the sustained release of at least one active principle (AP), characterized in that they comprise: a) a first polyelectrolyte polymer (PE1), preferably a linear alpha-polyamino acid, in a charged state, bearing hydrophobic groups (GH) on the side, said first polyelectrolyte polymer (PE1) forming spontaneously in water a colloidal solution of particles; at least a pHm value of the pH of between 3 and 8; b) a second polyelectrolyte polymer (PE2), preferably a linear alpha-polyamino acid, of opposite polarity to that of the first polyelectrolyte polymer (PE1), carrying hydrophobic groups (GH) on the side, said second polyelectrolyte polymer (PE2) forming in the water a solution or a colloidal solution at at least said pHm value of the pH; c) at least one active ingredient (PA) non-covalently associated with the particles of the colloidal solution of the first polyelectrolyte polymer (PEI); said particles for the sustained release of at least one active ingredient (PA) being obtained by mixing at a pH equal to pH m of the first polyelectrolyte polymer (PEI) in the form of a colloidal solution of particles associated with the active principle (PA) with the second polymer polyelectrolyte (PE2) in the form of a solution or colloidal solution.

The invention also relates to a method for preparing particles for the sustained release of at least one active principle (AP), these particles being in particular those described above, comprising the following steps: 1) the preparation, at a value pHm of the pH between 3 and 8, of an aqueous colloidal solution of a first polyelectrolyte polymer (PE1) in the charged state, carrying hydrophobic groups (GH) on the side, said first polyelectrolyte polymer (PEl) being capable of forming spontaneously in water a colloidal solution of particles with at least one pH value, pH of between 3 and 8;

2) adding at least one active ingredient (AP) to the first polyelectrolyte polymer (PEI) obtained in step 1, said active ingredient non-covalently associating with the particles of the colloidal solution of said first polyelectrolyte polymer ( PEI); 3) preparing a second polyelectrolyte polymer (PE2) of opposite polarity to that of the first polyelectrolyte polymer (PE1), carrying hydrophobic groups (GH) side, said second polymer polyelectrolyte (PE2) forming in water a solution or a colloidal solution at at least said pHm value of the pH; 4) mixing, at pH equal to pHm, the first polyelectrolyte polymer (PEI) in the form of a colloidal solution of particles to which the active ingredient (PA) obtained in step 2) is combined with the second polyelectrolyte polymer (PE2) under solution form or colloidal solution obtained in step 3). The invention also relates to a pharmaceutical formulation for the sustained release of at least one active ingredient (AP), said formulation comprising particles as described above. The invention also relates to a process for the preparation of medicaments, in particular for parenteral, mucosal, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral or tumoral, or even oral, nasal, pulmonary, vaginal, transdermal or ocular administration. , essentially consisting in implementing at least one formulation as described above.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the present description, a solution is understood to mean a homogeneous solvent and polymer mixture in the form of an individual chain.

In the present description, colloidal solution is understood to mean a suspension of particles whose average diameter measured by the T 'test is less than or equal to 0.5 μm.

In the present description, by half-neutralization pH is meant the pH at which half of the ionizable groups is ionized. In the present description, pHm means the pH at which the mixing of the first polyelectrolyte polymer (PEI) with which the active ingredient (PA) is associated with the second polyelectrolyte polymer (PE2).

In the present description, the physiological pH is defined as, for example, equal to 7.2 ± 0.4. In the present description, the term polyelectrolyte means a polymer carrying groups capable of ionizing in water, which creates a charge on the polymer.

In the present description, polyampholyte is understood to mean a polyelectrolyte carrying at least two types of groups which dissociate respectively into anionic and cationic groups. In the present description, the term "carrier" means that the group carried is pendant, that is to say that said group is a side group with respect to the main chain of the polymer. In particular, when the polymer is a polyamino acid comprising "amino acid" residues, said pendant group is a side group with respect to the "amino acid" residues and is a substituent of the γ carbonyl function of the "amino acid" residue which door.

In the present description, the polarity of a polyelectrolyte is understood to mean the polarity of the overall charge carried by this polyelectrolyte to the pHm value of the pH.

In the present description, by apparent density is meant the volume occupied by 1 g of particles. The apparent density is measured by any method known to those skilled in the art, such as the density gradient method.

In the present description, the term "small molecule" is understood to mean a molecule having a molecular weight of less than 1 kDa. To measure the size of the particles according to the invention, resulting from the combination of the first polyelectrolyte polymer (PEl) and the second polyelectrolyte polymer (PE2), the T test is used. To evaluate the particle size of the colloidal solution of the first polyelectrolyte polymer (PEI), the test T 'is preferably used.

The result of the test T is a median diameter D50, such that 50% of particles present in the sample have a size less than or equal to this value (D50). The result of the test T 'is a mean hydrodynamic diameter.

T-test for measuring the size of the microparticles by laser diffraction:

We obtain the data relating to the D50, which is the diameter below which 50% of the objects analyzed are found. This diameter of the particles according to the invention is measured according to the procedure defined below:

Particle solutions are prepared by diluting 400 μl of the sample to be analyzed in a 5 ml test tube with 600 μl of deionized water and then vortexing the preparation for 10 s (10 ± 5). These solutions are then introduced drop by drop into the measuring cell until the darkness is between

5% and 20%, then they are analyzed by light diffraction using a Malvern Mastersizer 2000 type device, operating at two wavelengths 466 and 632 nm.

The D50 of the particles is calculated from the Mie theory using the following refractive indices: n _flu , _of = 1.33 + i.0, npolymer ^≈ 1.59 + 10 and the Fraunhofer approximations, as described in ISO 13320 .

Test T 'measuring the size of the nanoparticles by quasi-elastic light scattering:

The average hydrodynamic diameter of the polymer particles according to the invention is measured according to the procedure Md defined below:

The polymer solutions are prepared at concentrations of 1 or 2 mg / ml in 0.15 M NaCl medium and left stirring for 24 h. These solutions are then filtered on 0.8-0.2 μm, before analyzing them in dynamic light scattering using a Malvern Compact Goniometer System, operating with a 632 wavelength He-Ne laser beam. , 8 nm and vertically polarized. The diameter The hydrodynamics of the polymer nanoparticles is calculated from the autocorrelation function of the electric field by the cumulant method, as described in "Surfactant Science Series" volume 22, Surfactant Solutions, Ed R. Zana, ch. 3, M. Dekker, 1984.

Test L for measuring the release of the active principle:

50 μl of formulation are injected into a cube of 1.5 cm of polyurethane-polyether foam (PU-PE) side bathed by a flow of 2.83 ml / h of an aqueous medium containing 30 mg / g of albumin bovine fraction V (Aldrich), 0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride (Aldrich PBS) and 0.015 M ammonium acetate (Aldrich). The continuous phase whose protein content is analyzed by ELISA (Immunotech kit IM3193) is regularly sampled.

The total mass of released protein can then be plotted by summing the one found in each of the samples and relate it to the total quantity injected.

For the purposes of the invention, the term "protein" refers to a protein as well as a peptide, whether it be an oligo or a polypeptide. This protein or peptide may or may not be modified, for example, by grafting one or more polyoxyethylene groups. The first and second polyelectrolyte polymers (PEl) and (PE2) are linear, biocompatible and biodegradable polymers bearing anionic and / or cationic ionizable groups, for example amine functions or carboxylic acids. Preferably, the polymer PE1 or PE2 carries ionizable groups of a given polarity (anionic or cationic). Such polymers are, for example, polyamino acids, anionic polysaccharides such as dextran sulfate, carboxymethylcellulose, gum arabic, hyaluronic acid and its derivatives, polygalacturonic compounds, polyglucuronic agents, or cationic polysaccharides such as chitosan, or also collagen and its gelatin derivatives. It is not excluded that a polymer bearing ionizable groups of a given polarity also carries a small fraction of 1 to 30 mol% of ionizable groups of the opposite polarity. In addition to anionic or cationic ionizable groups and hydrophobic groups, the first or second polyelectrolyte polymer (PE1) or (PE2) may optionally also carry non-ionizable groups, such as radicals chosen from a hydroxyethylamino radical, an alkylene glycol or a polyalkylene glycol; The net charge of the polymer depends on the pH value relative to the half-neutralization pH of the polymer. Thus, for a polyelectrolyte bearing carboxylic anionic functions, the net charge of the polymer will be close to zero at a pH of two units below the half-neutralization pH. Virtually all anionic functions will be ionized two pH units above the half-neutralization pH. Conversely, for a polymer carrying cationic functions, the net charge is canceled when the pH exceeds the half-neutralization pH by about two units.

In the present invention, the number of charges borne by the first or second polyelectrolyte polymer (PEl, PE2) under the conditions of mixing at the pHm value of the pH is obtained by the conventional method of acid-base titration:

A solution of polyelectrolyte concentrated at 2 mg / ml and containing 0.15 M of sodium chloride is brought to pH 3 by addition of 1 M acetic acid or 1 M sodium hydroxide. This solution is then titrated with a 0.05 M sodium hydroxide solution by recording the evolution of the pH as a function of the volume of sodium hydroxide added. The detection of the equivalence point (volume and pH), for example by the double-tangent method, makes it possible to detect the pH for which all the ionizable groups are ionized, ie where the degree of ionization is equal to 1. From this point, it is then possible to go back to the degree of ionization of the polyelectrolyte for any pH value. We can then define the pH of half-neutralization, that is to say the pH for which the degree of ionization is equal to 0.5. It is also possible to define the degree of ionization of the polyelectrolyte for the pHm value of the pH. In the particular case where the equivalence point is outside a pH range between 3 and 9, it is considered that all the ionizable groups are ionized over this pH range, that is to say that the degree of ionization is equal to 1 for pHs between 3 and 9.

Advantageously, the first and second polyelectrolyte polymers (PE1, PE2) may be linear alpha polyamino acids, recalling that if PE1 is a linear polyamino acid, PE2 is not polylysine or polyethyleneimine.

For the purposes of the invention and throughout the present disclosure, the term "polyamino acid" covers both natural polyamino acids and synthetic polyamino acids, as well as oligoamino acids comprising from 2 to 20 "amino acid" residues as well as polyamino acids comprising more than 20 residues "amino acid".

Preferably, the polyamino acids used in the present invention are oligomers or homopolymers comprising glutamic or aspartic acid repeating units or copolymers comprising a mixture of these two types of "amino acid" residues. The residues considered in these polymers are amino acids having the D or L or D / L configuration and are linked by their alpha or gamma positions for the glutamate or glutamic residue and alpha or beta for the aspartic or aspartate residue. The preferred "amino acid" residues of the main polyamino acid chain are those having the L-configuration and an alpha-type bond.

According to an even more preferred embodiment of the invention, the first and second polyelectrolyte polymers (PE1, PE2) are polyamino acids or a pharmaceutically acceptable salt thereof, the main chain of which is formed by residues chosen from group comprising aspartic residues, glutamic residues and combinations thereof, at least a portion of these residues being modified by grafting at least one hydrophobic group (GH) for at least the first polyelectrolyte polymer (PEI).

It is possible that the polymer PE2 also carries pendant hydrophobic groups.

According to a first variant, these polyamino acids are of the type of those described in patent application PCT WO-A-00/30618, according to which the hydrophobic groups

(GH) are identical to or different from one another and are selected from the group consisting of: (i) linear or branched, preferably linear, C 1 -C ₂₀ and even more preferably C ₂ -C ₁ alkyls, acyls or alkenyls; _S ;

(ii) hydrocarbon groups containing one or more heteroatoms, preferably those containing oxygen and / or sulfur and, more preferably, those of the following formula:

in which:

- _6O R is an alkyl, acyl or linear or branched alkenyl, preferably linear Cl -C ₂₀ and even more preferably C ₂ -C _S - R _6] and R ₆₂ are identical or different and represent with hydrogen or with a linear or branched, preferably linear C ₁ -C _{20 and} even more preferably C ₂ -C 15 _S alkyl, acyl or alkenyl radical, q = 1 to 100;

(iii) aryls, aralkyls or alkylaryls, preferably aryls; (iv) the hydrophobic derivatives, preferably the phosphatidylethanolamino radical or the radicals chosen from octyloxy-, dodecyloxy-, tetradecyloxy-, hexadecyloxy-, octadecyloxy-, 9-octadecenyloxy-, tocopheryloxy- or cholesteryloxy-.

For the purposes of the present invention, the term "hydrocarbon groups" means groups comprising, in particular, hydrogen and carbon atoms. Preferably, in this variant, the hydrophobic groups are selected from the following group of radicals: methyl, ethyl, propyl, docedyl, hexadecyl, octadecyl.

In a particularly preferred manner, the hydrophobic groups (GH) are chosen from the following group:

Linear or branched C 8 to C ₃₀ alkyls which may optionally comprise at least one unsaturation and / or at least one heteroatom,

C ₈ -C ₃₀ alkylaryls or arylalkyls which may optionally comprise at least one unsaturation and / or at least one heteroatom,

And the (poly) cyclic C ₈ to C ₃ o may optionally include at least one unsaturation and / or at least one heteroatom.

More precisely, at least one of the hydrophobic groups (GH) is obtained by grafting, starting from a precursor chosen from the group comprising: octanol, dodecanol, tetradecanol, hexadecanol, octadecanol, lec oleyl alcohol, tocopherol or cholesterol.

According to a variant of the invention, one of the polyelectrolyte polymers (PE1, PE2), or one of their pharmaceutically acceptable salts, corresponds to the following formula (I):

(I) in which:

R ¹ is H, C ₂ -C 10 linear alkyl, C ₃ -C 10 branched alkyl, benzyl, -R - [GH], or R together with NH is a terminal amino acid residue;

R is H, linear C ₂ -C ₁₀ acyl, branched C ₃ -C ₁₀ acyl, pyroglutamate or -R ⁴ - [GH];

R ⁴ represents a direct bond or a "spacer" based on 1 to 4 amino acid residues;

A ¹ and A ² independently represent -CH ₂ - (aspartic residue) or -CH ₂ -CH ₂ - (glutamic residue); N / (n + m) is defined as the molar grafting level and its value is sufficiently low for the polymer dissolved in water at pH 7 and at 25 ^° C. to form a colloidal suspension of polymer particles. ;

N + m varies from 10 to 1000, preferably from 50 to 300;

GH represents a hydrophobic group comprising 6 to 30 carbon atoms or is selected from the group comprising the radicals as defined above in paragraphs (i), (ii), (iii) and (iv).

For more details on the preparation and synthesis of polyamino acids of formula (I), reference is made to the patent applications FR 02 07008 and FR 03 50190.

According to another possibility, the polyelectrolyte polymer PE 2, or one of its pharmaceutically acceptable salts, corresponds to one of the following formulas (II), (III) and (IV):

in which :

GH represents a hydrophobic group having 6 to 30 carbon atoms;

R ³⁰ is a linear C ₂ -C ₆ alkyl group; R ⁵⁰ is alkyl, dialkoxy or C ₂ -C ₆ diamino; R ⁴ represents a direct bond or a "spacer" based on 1 to 4 amino acid residues;

A ¹ and A ² independently represent a radical -CH ₂ - (aspartic residue) or -CH ₂ -CH ₂ - (glutamic residue); • n '+ m' or n "is defined as the degree of polymerization and varies from 10 to

1000, preferably between 50 and 300.

For more details on the preparation and synthesis of polyamino acids of formula (I), (II) and (IV), reference will be made to the patent application FR 03 50641.

According to another possibility, one of the polyelectrolyte polymers (PE1, PE2), or one of their pharmaceutically acceptable salts, corresponds to the following formula (V):

in which :

"E- independently represents:

an -NHR in which R represents H, a linear C ₂ -C ₁₀ alkyl, a branched C ₃ -C ₁₀ alkyl or a benzyl, an amino acid residue or a terminal amino acid derivative of formula:

H ₇

-NH - C COR ⁷

in which

R ⁷ is OH, OR ⁹ or NHR ¹⁰ , and R ⁸ , R ⁹ and R ¹ independently represent H, linear alkyl in

C ₂ to C ₁₀ , branched C ₃ to C ₁₀ alkyl or benzyl; B is a direct bond or a divalent, trivalent or tetravalent linking group, preferably selected from the following radicals:

-O-, -NH-, - N (-Ci- ₅ alkyl) -, an amino acid residue (preferably natural), diol, triol, diamine, triamine, amino alcohol or hydroxy having 1 to 6 carbon atoms, D is H, linear C ₂ -C ₁₀ acyl, branched C ₃ -C ₁₀ acyl, or pyroglutamate;

GH represents a hydrophobic group having 6 to 30 carbon atoms; R ⁷⁰ represents a radical chosen from the following group:

-NH- (CH ₂ ) _W -NH ₃ ⁺ with w between 2 and 6, and preferably w is equal to 4,

- -NH- (CH ₂ ) ₄ -NH-C (= NH) -NH ₃ ⁺ ,

- -O- (CH ₂ ) ₂ -NH ₃ ⁺ ,

-O- (CH ₂ ) ₂ -N ⁺ (CH ₃ ) ₃ ,

or

an alkyl ester (preferably -COOMe and -COOEt), -CH ₂ OH, -Q = O) -NH ₂ , -C (= O) -NH-CH ₃ or -C (= O) -N ( CH ₃ ) ₂ ; an amino acid residue or amino acid derivative of formula:

in which :

X is an oxygen atom or -NH-,

R ¹ is H, linear C ₂ -C ₁₀ alkyl, branched C ₃ -C ₁₀ alkyl or benzyl,

-R ¹³ is - (CH ₂ ) ₄ NH ₃ ⁺ , - (CH ₂ ) ₃ -NH-C (= NH) -NH ₃ ⁺ , - (CH ₂ ) ₃ NH ₃ ⁺ ; the counteranion of R ⁷⁰ is a chloride, a sulfate, a phosphate or an acetate, preferably a chloride;

R represents a hydroxyethylamino-, an alkylene glycol residue or a polyoxyalkylene; • p, q, r and s are positive integers;

(P) / (p + q + r + s) is defined as the molar grafting ratio of the hydrophobic groups GH varies from 2 to 99 mol%, and preferably between 5 and 50%, provided that each copolymer chain has on average at least 3 hydrophobic grafts;

• (q) / (p + q + r + s) is defined as the molar grafting rate of the cationic groups and varies from 1 to 99 mol%;

"(p + q + r + s) varies from 10 to 1000, preferably from 30 to 500;

• (r) / (p + q + r + s) varies from 0 to 98 mol%; (s) / (p + q + r + s) varies from 0 to 98 mol%.

The derivatives of lysine, ornithine, and arginine may be, for example, ethyl and methyl esters, amides, and methylated amides.

Preferably according to this variant, the hydrophobic groups GH and the cationic groups are randomly arranged in pendant groups.

It is also preferable that the hydrophobic mole ratio of the polyglutamate is between 2 and 99%, and preferably between 5 and 50% provided that each polymer chain has on average at least 3 hydrophobic grafts. The ratio (q) / (p + q + r + s) of the polyglutamates means that they may contain from 1 to about 97 mole percent of cationic charge containing groups.

The ratio (s) / (p + q + r + s) of polyglutamates means that they can be anionic, neutral or cationic at neutral pH.

For more details on the preparation and synthesis of polyamino acids of formula

(V) derived from histidine, reference is made to the patent application FR 05 53302. For more details on the preparation and synthesis of polyamino acids of formula (V) other than those derived from histidine, reference will be made usefully at the patent application FR 07 03185.

According to an interesting variant, the group R ⁴ or B represented in the preceding formulas represents a direct bond.

According to another possibility, one of the polyelectrolyte polymers (PE1, PE2) comprises hydroxyalkyl (preferably ethyl) glutamine residues and a multiplicity of hydrophobic groups (GH) pendant and identical or different from each other. The hydroxyalkylglutamine residues also carry hydroxyalkylamine groups. These hydroxyalkylamine groups are preferably bonded to the copolymer via an amide bond. The hydroxyalkylamine groups that can be used to functionalize the glutamate residues of these hydroxyalkylglutamine residues are identical or different from each other and are for example chosen from the following groups: 2-hydroxyethylamino, 3-hydroxypropylamino, 2,3-dihydroxypropylamino, tris (hydroxymethyl) methylamino and 6-hydroxyhexylamino. Advantageously, at least one of the hydrophobic groups GH used in the present invention is included in a hydrophobic graft comprising at least one spacer (or "spacer") to connect the hydrophobic group GH to a chain of copolyglutamates (eg a main chain - skeleton-copolyglutamates). This patella may comprise, eg at least one direct covalent bond and / or at least one amide bond and / or at least one ester bond. For example, the patella may be of the type belonging to the group comprising in particular: the "amino acid" residues different from the constituent monomeric unit of the copolyglutamate, the aminoalcohol derivatives, the polyamine derivatives (for example the diamines), the derivatives of polyols (for example diols) and derivatives of hydroxy acids. The grafting of the GH on the copolyglutamate or polyalkylglutamine chain may involve the use of GH precursors that are capable of binding to the copolyglutamate chain or the hydroxyalkylglutamine residues. The precursors of GH are, in practice and without being limited to, selected from the group comprising alcohols and amines, these compounds being easily functionalized by those skilled in the art. For more details on these hydroxyalkyl (preferably hydroxyethyl) glutamine residues, reference is made to FR 2,881,140.

According to an advantageous variant, especially according to at least one of the various possibilities mentioned above, all or part of the hydrophobic groups (GH) used in the present invention are independently selected from the group of radicals comprising:

"a linear or branched alkoxy having from 6 to 30 carbon atoms and which may comprise at least one heteroatom (preferably O and / or N and / or S) and / or at least one unsaturation,

An alkoxy having 6 to 30 carbon atoms and having one or more annealed carbocycles and optionally containing at least one unsaturation and / or at least one heteroatom (preferably O and / or N and / or S), an alkoxyaryl or a aryloxyalkyl of 7 to 30 carbon atoms and may comprise at least one unsaturation and / or at least one heteroatom (preferably O and / or N and / or S).

According to another advantageous variant, in particular according to at least one of the various abovementioned possibilities, the hydrophobic group (GH) is derived from an alcoholic precursor chosen from the group comprising: octanol, dodecanol, tetradecanol, hexadecanol, octadecanol, oleyl alcohol, tocopherol or cholesterol, and R represents a direct linkage.

According to another advantageous variant, the hydrophobic groups (GH), in particular according to at least one of the various abovementioned possibilities, each independently represent a monovalent radical of the following formula:

(GH) in which:

R represents a methyl (alanine), isopropyl (valine), isobutyl (leucine), secbutyl (isoleucine), benzyl (phenylalanine);

- R ⁶ represents a hydrophobic radical containing from 6 to 30 carbon atoms;

- 1 varies from 0 to 6.

According to a remarkable feature of the invention, all or part of the hydrophobic radicals R ⁶ of the hydrophobic groups (GH) are independently selected from the group of radicals comprising:

A linear or branched alkoxy having from 6 to 30 carbon atoms and which may comprise at least one heteroatom (preferably O and / or N and / or S) and / or at least one unsaturation, an alkoxy having 6 to 30 atoms of carbon and having one or more annealed carbocycles and optionally containing at least one unsaturation and / or at least one heteroatom (preferably O and / or N and / or S),

An alkoxyaryl or an aryloxyalkyl of 7 to 30 carbon atoms and which may comprise at least one unsaturation and / or at least one hetero atom (preferably O and / or N and / or S).

In practice and without being limiting, said hydrophobic radical R ⁶ is derived from an alcoholic precursor chosen from the group comprising: octanol, dodecanol, tetradecanol, hexadecanol, octadecanol, oleyl alcohol, tocopherol or cholesterol.

Advantageously, the main chain of the polyelectrolyte polyamino acids (PE1, PE2) used in the invention is chosen from the group comprising alpha-L-glutamate homopolymers, alpha-L-glutamic homopolymers, alpha-L-glutamic homopolymers and L-aspartate, alpha-L-aspartic homopolymers, alpha-L-aspartate / alpha-L-glutamate copolymers and alpha-L-aspartic / alpha-L-glutamic copolymers. Remarkably, the distribution of the aspartic and / or glutamic residues of the main polyamino acid chain of the polyelectrolyte polymer PE1 or PE2 is such that the polymer thus formed is either random, either of the block type or of the multiblock type.

According to another mode of definition, the polyelectrolyte polymer PE1 or PE2 has a molar mass which is between 2,000 and 100,000 g / mol, and preferably between 5,000 and 40,000 g / mol.

The degree of polymerization of the first and second polyelectrolyte polymers (PE1, PE2) is between 50 and 500, preferably between 70 and 300.

The mole fraction of the chain members of the main chain substituted with hydrophobic groups is between 1 and 40 mol%, preferably between 3 and 30 mol%.

The polymers used in the present invention are chosen from the various families described above so that they are globally cationic or anionic at the pH value equal to pHm. An essential characteristic of the first polyelectrolyte polymer (PEl) bearing lateral hydrophobic groups is to be able to spontaneously form in water a colloidal solution.

Without wishing to be bound by theory, it can be argued that the supramolecular association of hydrophobic groups to form hydrophobic domains leads to the formation of nanoparticles. Each nanoparticle consists of one or more polymer chains PEl more or less condensed around its hydrophobic domains. It should be understood that the polymers used in the invention contain ionizable functions which are, depending on the pH and the composition, either neutral

(For example -COOH, -NH ₂ ), or ionized (for example -COO ^" , -NH ₃ ⁺ ), for this reason, the solubility in an aqueous phase is directly a function of the rate of ionized functions and therefore of the pH. aqueous solution, in the case of carboxylic functions, the counter-ion may be a metal cation such as sodium, calcium or magnesium, or an organic cation such as triethanolamine, tris (hydroxymethyl) -aminomethane or a polyamine such as The counteranion of the cationic groups is preferably selected from the group comprising a chloride, a sulfate, a phosphate or an acetate.

Knowing the pH of half-neutralization, one skilled in the art knows how to adjust the pH so that the degree of ionization of the polymer is sufficiently high and ensures the stability of the colloidal solution. The polyelectrolytes of the polyamino acid type which can be used in the present invention are obtained, for example, by methods known to those skilled in the art. The random polyamino acids can be obtained by grafting the hydrophobic graft, previously functionalized by the "spacer", directly onto the polymer. by a conventional coupling reaction. The block or multiblock polyamino acid polyelectrolytes can be obtained by sequential polymerization of the corresponding N-carboxy-amino acid anhydrides (NCA).

For example, a polyamino acid, homopolyglutamate, homopolyaspartate or a glutamate / aspartate, block, multiblock or random copolymer is prepared according to conventional methods.

For obtaining alpha-type polyamino acid, the most common technique is based on the polymerization of N-carboxy-amino acid anhydrides (NCA), described, for example, in the article "Biopolymers, 1976, 15, 1869 and in HR Kricheldorf's "Alpha-Aminoacid-N-Carboxy Anhydrides and related Heterocycles" Springer Verlag (1987) NCA derivatives are preferably NCA-O-Me, NCA-O-Et or NCA-derivatives. O-Bz (Me = Methyl, Et = Ethyl and Bz = Benzyl) The polymers are then hydrolyzed under appropriate conditions to obtain the polymer in its acid form These methods are inspired by the description given in patent FR 2 801 226 A number of usable polymers according to the invention, for example, of the poly (alpha-L-aspartic), poly (alpha-L-glutamic), poly (alpha-D-glutamic) and poly (gamma) type can be used. -L-glutamic) of variable masses are available commercially.The polyaspartic alpha-beta type is obtained p condensation of aspartic acid (to obtain polysuccinimide) followed by basic hydrolysis (cf. Tomida et al. Polymer 1997, 38, 4733-36).

The coupling of the hydrophobic graft GH with an acidic function of the polymer is easily achieved by reacting the polyamino acid in the presence of a carbodiimide as a coupling agent and optionally a catalyst such as 4-dimethylaminopyridine and in a suitable solvent such as dimethylformamide ( DMF), N-methylpyrrolidone (NMP) or dimethylsulfoxide (DMSO). The carbodiimide is, for example, dicyclohexylcarbodiimide or diisopropylcarbodiimide. The degree of grafting is chemically controlled by the stoichiometry of the constituents and reactants or the reaction time. Hydrophobic grafts functionalized by a "spacer" are obtained by conventional peptide coupling or by direct condensation by acid catalysis. The coupling of the cationic and optionally neutral groups with an acid function of the polymer is carried out simultaneously in a second step in the presence of a chloroformate as coupling agent and in a suitable solvent such as dimethylformamide, N-methylpyrrolidone (NMP) or dimethylsulfoxide (DMSO).

In the case where the cationic group contains two chemically undifferentiated amino functions (eg linear diamine), it can be introduced in a form in which one of the two functions is protected. A last step of cleavage of the protecting group is then added. The polymerization chemistry and coupling reactions of the groups are conventional and well known to those skilled in the art (see for example the patents or patent applications of the applicant mentioned above).

For the synthesis of block or multiblock copolymer, NCA derivatives previously synthesized with the hydrophobic graft are used. For example, the NCA-hydrophobic derivative is copolymerized with the NCA-O-Benzyl and then the benzyl groups are selectively removed by hydrolysis.

Examples of particularly preferred combinations of polyelectrolyte polymers (PE1 and PE2) according to the invention are described in the examples hereinafter.

An essential characteristic of the polymers used in the invention is that the first polyelectrolyte polymer (PE1) is in the form of a colloidal solution, and that the second polyelectrolyte polymer (PE2) is in the form of a solution or colloidal solution for at least one value, pHm, the pH between 3 and 8. In order to fulfill this condition, the half-neutralization pH of the cationic polymer will be sufficiently high, for example greater than 5.5, preferably greater than 6, or even greater than 8; and the half-neutralization pH of the anionic polymer will be sufficiently low, for example less than 6.5, preferably less than 6.0 or even less than 5.5. More particularly, in a variant in which the first polyelectrolyte polymer (PEI) is anionic, the latter is chosen so that it has a half-neutralization pH of between 3 and 6.5, and preferably between 4.5 and 6.5. According to this variant, the first polyelectrolyte polymer (PE1) forms a colloidal solution for a pHm value of the pH of between 6 and 8. Such a PEl polymer is described in particular in Example 1a).

In this case, and according to a first possibility, the second electrolyte polymer (PE2) is cationic and forms a colloidal solution with a pH of less than 8. Preferably, the second polyelectrolyte polymer (PE2) is chosen so that its pH of half neutralization is greater than 8. Such a polymer PE2 is in particular described in Example Id). Thus, according to this first possibility, the first polyelectrolyte polymer (PE1) forms a colloidal solution and the second polyelectrolyte polymer (PE2) forms a solution or a colloidal solution for the pHm value of the pH between 6 and 8.

In this case, according to an important characteristic of the invention, the ratio of the mass of the first polyelectrolyte polymer (PEl) to the mass of the second polyelectrolyte polymer (PE2) is chosen so that the load ratio Z, ratio of the number of moles cationic ionized groups to the number of moles of anionic ionized groups, measured at pHm, between 0.25 and 3, and preferably between 0.25 and 1.5. According to a second possibility, the second polyelectrolyte polymer (PE2) is cationic and forms a colloidal solution with a pH below 6 and a precipitate with a pH greater than 6.5. Preferably, the second polyelectrolyte polymer (PE2) is chosen so that its pH of half-neutralization is between 5.5 and 7. Such a polymer PE2 is described in particular in Example Ic). In this case, according to an important characteristic of the invention, the first polyelectrolyte polymer (PE1) forms a colloidal solution and the second polyelectrolyte polymer (PE2) forms a solution or a colloidal solution for a pH value, pHm, of between 3 and 6. The ratio of the mass of the first polyelectrolyte polymer (PEl) to the mass of the second polyelectrolyte polymer (P E2) is chosen so that the charge ratio Z, measured at pHm, is between 3.5 and 30, of preferably between 5 and 15, and even more preferably between 8 and 12.

In another variant in which the first polyelectrolyte polymer (PEI) is cationic, the latter is chosen so that its pH of half-neutralization is greater than 5. In this case, the second electrolyte polymer (PE2) is anionic and is chosen so that its pH of half-neutralization is between 3 and 6.5, and preferably between

4.5 and 6.5.

The particles according to the invention have, at physiological pH, a size, measured in a T test, of between 1 and 100 μm.

Advantageously, the particles according to the invention are not chemically crosslinked.

In a particular embodiment of the invention, the particles have, at physiological pH, an apparent density in high polymer, of between 0.15 and 1.1, preferably of between 0.3 and 1.0, and even more preferentially between 0.5 and 1.0. A high polymer density reflects the existence within the particles of a dense network of polymer chains. Without wishing to be bound by the theory, it can be supposed that this dense network slows the diffusion of the active ingredient (PA) contained in the particles according to the invention to the external environment and thus contributes to slowing down its release. A surprising aspect of the dense particles according to the invention is that the network of polymer chains which constitutes them makes it possible to slow the release of the AP without trapping this same PA in the heart of the particles. Thus, the carrier according to the invention makes it possible to obtain both prolonged release of the AP and good bioavailability.

In some cases, and in particular in the case of peptides or proteins having a high affinity with the microparticles according to the invention, it may be advantageous to modulate the release rate of the active ingredient, to accelerate the release, and / or to improve its bioavailability. After numerous tests, it has been demonstrated by the Applicant that the release of the protein or peptide can be facilitated when the polymer PE1 or PE2, of a given polarity, is also a carrier of ionizable groups of the opposite polarity and / or nonionizable groups such as groups substituted by a hydroxyethylamino radical.

Thus, in a particular embodiment of the invention, one of the two PEl or PE2 polymers simultaneously comprises: from 15 to 50 mol% of glutamate monomers;

from 20 to 55 mol% of nonionizable monomers such as groups substituted with a hydroxyethylamino radical; from 10 to 40 mol% of monomers carrying cationic groups of neutralization pH greater than 8; from 3 to 15 mol% of nonionizable monomers substituted with a hydrophobic group.

In another particular embodiment of the invention, the polymer PE1 or PE2 is cationic and simultaneously comprises: from 0 to 5 mol% of glutamate monomers; from 50 to 85 mol% of nonionizable monomers such as groups substituted with a hydroxyethylamino radical; from 10 to 40 mol% of monomers carrying cationic groups of neutralization pH greater than 8; from 3 to 15 mol% of nonionizable monomers substituted with a hydrophobic group.

In a particular embodiment of the invention, the total concentration of polymer (PE1 + PE2) contained in the formulation is between 4 and 15 mg / ml, especially when the active ingredient is a therapeutic protein. In this concentration range, the formulation is easily injectable by a small diameter needle, for example a Gauge 27, even 29, and even 31 needle. Examples 3 and 4 describe in detail such formulations.

Regarding the active principle, it is preferably chosen from the group comprising: proteins, glycoproteins, proteins linked to one or more polyalkylene glycol chains [preferably polyethylene glycol (PEG): "PEGylated proteins"], peptides, polysaccharides liposaccharides, oligonucleotides, polynucleotides and mixtures thereof, and more preferably still in the erythropro-retin subgroup, such as epoetin alpha, epoetin beta, darbepoetin, hemoglobin, and the like. or their derivatives; oxytocin, vasopressin, adrenocorticotropic hormone, epidermal growth factor, platelet growth factor (PDGF), stimulatory factors of hematopoiesis and mixtures thereof, blood factors, such as alteplase, tenecteplase, factor VΙI (a), factor VII; hemoglobin, cytochromes, prolactin albumins, luliberin (luteinizing hormone releasing hormone or LHRH) or the like, such as leuprolide, goserelin, triptorelin, buserelin, nafarelin; LHRH antagonists, competitors of LHRH, human growth hormones (GH), porcine or bovine hormones, growth hormone releasing hormone, insulin, somatostatin, glucagon, interleukins or their mixtures (IL-2, IL-1, IL-12), interferons, such as interferon alpha, alpha-2b, beta, beta, or γ; gastrin, tetragastrin, pentagastrin, urogastrone, secretin, calcitonin, enkephalins, endomorphins, angiotensins, thyrotropin releasing factor (TRH), tumor necrosis factor (TNF), growth factors (NGF), growth factors such as beclapermine, trafermine, ancestime, keratinocyte growth factor, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) ), macrophage colony stimulating factor (M-CSF), heparinase, bone morphogenetic protein (BMP), hANP, glucagon-like peptide (GLP-I), VEG-F, recombinant hepatitis B (rHBsAg), renin, cytokines, bradykinin, bacitracins, polymixins, colistins, tyrocidine, gramicidines, etanercept, imiglucerase, alpha drotrecogin, cyclosporins and synthetic analogies, the modifications and pharmaceutically active fragments of enzymes, cytokines, antibodies, antigens and vaccines, antibodies such as rituximab, infixamab, trastuzumab, adalimumab, omalizumab, tositumomab, efalizumab, and cetuximab. Other active ingredients are polysaccharides (eg, heparin) and oligo- or polynucleotides, DNA, RNA, iRNA, antibiotics, and living cells. Another class of active ingredients includes pharmaceutical substances acting on the central nervous system, for example, risperidone, zuclopenthixol, fluphenazine, perphenazine, flupentixol, haloperidol, fluspirilene, quetiapine, clozapine, amisulprid, sulpirid, ziprasidone, etc.

According to one variant, the active ingredient is a small hydrophobic, hydrophilic or amphiphilic organic molecule of the type belonging to the family of anthracyclines, taxoids or camptothecins or of the type belonging to the family of peptides such as leuprolide or cyclosporin and mixtures thereof. As used herein, a small molecule is especially a small non-protein molecule, for example, free of amino acids.

According to another variant, the active ingredient is advantageously chosen from at least one of the following families of active substances: alcohol abuse treatment agents, agents for treating Alzheimer's disease, anesthetics, Pacromegaly treatment agents, analgesics, antiasthmatics, allergy treatment agents, anticancer agents, anti-inflammatories, anticoagulants and antithrombotics, anticonvulsants, anti-epileptics, antidiabetics, antiemetics, antiglaucoma , antihistamines, antihistamines infections, antibiotics, antifungals, antivirals, antiparkinsonians, anti-cholinergics, antitussives, carbonic anhydrase inhibitors, cardiovascular agents, lipid-lowering agents, anti-arrhythmics, vasodilators, anti-angines, antihypertensives , vasoprotectants, cholinesterase inhibitors, central nervous system treatment agents, central nervous system stimulants, contraceptives, fertility promoters, uterine labor inducers and inhibitors, cystic fibrosis agents , dopamine receptor agonists, endometriosis agents, erectile dysfunction agents, fertility agents, gastrointestinal treatment agents, immunomodulators and immunosuppressants, the agents for treating memory disorders, anti-migraine patients, muscle relaxants, nucleoside analogues, osteoporosis agents, parasympathomimetics, prostaglandins, psychotherapeutic agents, sedatives, hypnotics and tranquillizers, neuroleptics, anxiolytics, psychostimulants, antidepressants, dermatological treatment agents, steroids and hormones, amphetamines, anorexics, non-painkillers, anti-epileptics, barbiturates, benzodiazepines, hypnotics, laxatives, psychotropics and all combinations thereof . According to another of its aspects, the subject of the invention is a process for preparing particles for the sustained release of at least one active principle, these particles being in particular those described above, said process comprising the following steps:

1) the preparation, at a pHm value of pH between 3 and 8, of an aqueous colloidal solution of a first polyelectrolyte polymer (PE1) in the charged state, carrying hydrophobic groups (GH) on the side, said first polymer polyelectrolyte (PE1) being capable of spontaneously forming in water a colloidal solution of particles with at least a pH value, pHm, of pH between 3 and 8;

2) adding at least one active ingredient (AP) to the first polyelectrolyte polymer (PEI) obtained in step 1, said active ingredient non-covalently associating with the particles of the colloidal solution of said first polyelectrolyte polymer ( PEI);

3) preparing a second polyelectrolyte polymer (PE2) of opposite polarity to that of the first polyelectrolyte polymer (PE1), said second polyelectrolyte polymer (PE2) forming in water a solution or a colloidal solution with at least said value, pHm, pH; provided that, if the first electrolyte polymer (PE1) is a polyamino acid, then the second polyelectrolyte polymer (PE2) is neither polylysine nor polyethylene imine;

4) the mixture at a pH equal to pH m of the first polyelectrolyte polymer (PEI) in the form of a colloidal solution of particles to which the active ingredient (PA) obtained in step 2) is associated with the second polyelectrolyte polymer (PE2) in the form of solution or colloidal solution obtained in step 3).

The subject of the invention is also a method for preparing particles for the sustained release of at least one active principle (AP), these particles corresponding in particular to certain ones described above, comprising the following steps:

1) the preparation, at a pHm value of pH between 3 and 8, of an aqueous colloidal solution of a first polyelectrolyte polymer (PE1) in the charged state, carrying hydrophobic groups (GH) on the side, said first polymer polyelectrolyte (PE1) being capable of spontaneously forming in water a colloidal solution of particles with at least a pH value, pHm, of pH between 3 and 8;

2) adding at least one active ingredient (AP) to the first polyelectrolyte polymer (PEI) obtained in step 1, said active ingredient non-covalently associating with the particles of the colloidal solution of said first polyelectrolyte polymer ( PEI);

3) the preparation of a second polyelectrolyte polymer (P E2) of opposite polarity to that of the first polyelectrolyte polymer (PE1) carrying hydrophobic groups (GH) on the side, said second polyelectrolyte polymer (PE2) forming a solution in water; or a colloidal solution at at least said pHm value of the pH;

4) the mixture at a pH equal to pH m of the first polyelectrolyte polymer (PEI) in the form of a colloidal solution of particles to which the active ingredient (PA) obtained in step 2) is associated with the second polyelectrolyte polymer (PE2) in the form of solution or colloidal solution obtained in step 3).

An essential characteristic of the process according to the invention is to form particles, spontaneously, by simple mixing at pH m of a colloidal solution of particles of the first polyelectrolyte polymer (PEI) loaded with active ingredient (PA) and a solution or d a colloidal solution of the second polyelectrolyte polymer (PE2) of opposite polarity.

The active ingredients such as proteins, peptides or small molecules can spontaneously associate with the first polymer (PE1) of polyamino acid type. The loading the nanoparticles of the first polyelectrolyte polymer (PEI) with the active ingredient (PA) is carried out by simple mixing of a solution of active principle (PA) with a colloidal solution of the first polyelectrolyte polymer (PEI). This association is purely physical and does not imply the creation of a co-valent bond between the active ingredient (PA) and the polymer (PEI). Without being bound by theory, it can be assumed that this non-specific association is effected by hydrophobic and / or electrostatic interaction between the polymer (PEl) and the active ingredient (PA). It should be noted that it is not necessary, and often even undesirable, to bind the PA to the nanoparticles of PEI by specific receptors of a peptide nature or of the antigen / antibody or enzyme / substrate type. In a preferred embodiment of the method according to the invention, there is no provision for a chemical crosslinking step of the particles obtained. The particles according to the invention are therefore not chemically crosslinked and nevertheless release the active ingredient (PA) over a prolonged period. This absence of chemical crosslinking is a decisive advantage of the particles according to the invention. Indeed, the absence of chemical crosslinking makes it possible to avoid the chemical degradation of the active ingredient (PA) during the step of crosslinking the particles containing the active ingredient (PA). Such chemical crosslinking is in fact generally carried out by activations of polymerizable entities and involves potentially denaturing agents such as UV radiation or glutaraldehyde.

Advantageously, the process according to the invention comprises a step of dehydration of the suspension of particles obtained (for example by lyophilization or atomization), in order to obtain them in the form of a dry powder.

According to another of its aspects, the subject of the invention is a pharmaceutical formulation for the sustained release of at least one active ingredient (AP), comprising an aqueous suspension of particles as described above or those obtained by the process described above.

The present invention also relates to a solid pharmaceutical formulation for the sustained release of at least one active ingredient (AP), comprising a dry powder form:

• based on particles containing at least one active ingredient (AP), these particles being those described above or those obtained by the method described above;

Or obtained from the formulation comprising a suspension in aqueous solution mentioned above.

Advantageously, such a solid pharmaceutical formulation is used for inhalation and pulmonary administration. According to another of its aspects, the subject of the invention is a process for preparing medicaments, in particular for parenteral, mucosal, subcutaneous, intramuscular, intradermal, transdermal, intraperitoneal, intracerebral or tumor administration, or even orally. , nasal, pulmonary, vaginal or ocular, said method essentially consisting in implementing at least one of the formulations described above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: in vitro release of IFN-α from the particle formulations of Example 2 (white circles), Example 3.1 (black triangles), Example 3.2 (black diamonds), the Example 3.3 (black squares), Example 4 (black circles) and Example 5 (lines).

EXAMPLES

1) Summaries:

a) Synthesis of a polyelectrolyte polymer PEl-A bearing hydrophobic groups, anionic (polyglutamate grafted with alpha-tocopherol of synthetic origin)

15 g of an alpha-L-polyglutamic acid (mass equivalent to about 16900 Da) are solubilized with respect to a polyoxyethylene standard and obtained by polymerization of NCA-GIuOMe followed by hydrolysis as described in the FR-patent application. A-2 801 226) in 288 ml of dimethylformamide (DMF) by heating at 80 ⁰ C until the polymer is solubilized. The solution is cooled to 15 ^° C. and 2.5 g of D, L-alpha-tocopherol (> 98% obtained from Fluka®) preliminarily solubilized in 8 ml of DMF, 280 mg of 4-dimethylaminopyridine, which has been solubilized beforehand, is added successively. 1 ml of DMF and 1.6 g of diisopropylcarbodiimide previously solubilized in 6 ml of DMF. After stirring for 3 h, the reaction medium is poured into 1200 ml of water containing 15% of sodium chloride and hydrochloric acid (pH = 2). The precipitated polymer is then recovered by filtration, washed with 0.1 N hydrochloric acid, water and with diisopropyl ether. The polymer is then dried in an oven under vacuum at 40 ^° C. A yield of the order of 90% is obtained. The molar mass measured by size exclusion chromatography is 15500 relative to a polyoxyethylene standard. The grafted tocopherol level, estimated by proton NMR spectroscopy, is 5.1 mol%.

b) Synthesis of anionic PEl-B polyelectrolyte polymer (sodium polyglutamate)

The synthesis of an alpha-L-polyglutamic acid is adapted as described in patent application FR-A-2 801 226. The molar mass measured by size exclusion chromatography is 16900 Da relative to a polyoxyethylene standard.

c) Synthesis of a cationic PE2-A polyelectrolyte polymer (polyglutamate grafted with alpha-tocopherol of synthetic origin and with histidinamide)

Indices and groups: m = 11, p = 209, q = 0, T = D, L-alpha-tocopheryl (T)

3 g of a poly (glutamic acid) of DP 220 randomly grafted to 5% with racemic alpha-tocopherol are solubilized by heating at 80 ^° C. in 38 ml of NMP. To this cooled solution at 0 ^° C. are added 2.74 g of isobutyl chloroformate then 2.2 ml of N-methyl morpholine. The reaction medium is stirred for 10 min while maintaining the temperature at 0 ^° C. In parallel, 8.65 g of histidinamide dihydrochloride are suspended in 108 ml of NMP. 10.6 ml of triethylamine are then added and the suspension obtained is stirred at 20 ^° C. for a few minutes and then cooled to 0 ^° C. The solution of activated polymer is then added to the suspension of histidinamide. The reaction medium is stirred for 2 h at 0 ^° C. and then overnight at 20 ^° C. 0.62 ml of 35% HCl are then added, followed by 83 ml of water. The solution obtained is then poured into 500 ml of water at a pH of between 3 and 4. The solution is then diafiltered against 8 volumes of salt water (0.9% NaCl) and 4 volumes of water. The polymer solution is then concentrated to a volume of 300 mL (the polymer concentration is 18 mg / g). The percentage of grafted histidinamide determined by ¹ H NMR in D ₂ O is 95%.

d) Synthesis of a cationic PE2-B polyelectrolyte polymer (polyglutamate grafted with alpha-tocopherol of synthetic origin and with arginine)

Indices and groups: T = D, L-α-tocopheryl, p = s = 11, q = 198, r = O

Ten grams of a poly (glutamic acid) of DP 220 grafted at 5% randomly with racemic α-tocopherol are solubilized in 125 mL of NMP at 80 ^° C. This solution is cooled to 0 ^° C., and 8.7 ml of chlor-butyl chloroformate and then 7.35 ml of N-methyl morpholine are added. This reaction mixture is stirred for 15 minutes at 0 ^° C. In parallel, 24.67 g of argininamide dihydrochloride are suspended in 308 ml of NMP and 14.7 ml of triethylamine are added. The suspension obtained is stirred for a few minutes at 20 ^° C. and then cooled to 0 ^° C. The milky suspension of activated polymer is then added to this suspension, and the reaction mixture is stirred for 2 h at 0 ^° C. and then overnight at 20 ^° C. ⁰ C. After addition of 2.1 mL of a solution of 35% HCl then 10O mL of water, the reaction mixture was poured dropwise into 1.6 L of water. The solution obtained is diafiltered against 8 volumes of salt water (0.9%) then 4 volumes of water, and concentrated to a volume of about 250 mL. The percentage of grafted argininamide, determined by proton NMR in D ₂ O, is 90%. e) Synthesis of a cationic PE2-C polyelectrolyte polymer (polyglutamate grafted with alpha-tocopherol of synthetic origin, arginine and ethanolamine)

Indices and groups: T = D, L-α-tocopherol, p = 11, q = 88, r = 99, s = 22

Ten grams of a poly (glutamic acid) of DP 220 grafted at 5% randomly with racemic α-tocopherol are solubilized in 125 mL of NMP at 80 ^° C. This solution is cooled to 0 ^° C., and 9.1 ml of tert-butyl chloroformate and then 7.71 ml of N-methyl morpholine are added. This reaction mixture is stirred for 15 min at 0 ^° C. In parallel, 8.2 g of argininamide dihydrochloride are suspended in 103 mL of NMP and 9.31 mL of triethylamine are added. Another 1.6 ml of ethanolamine is added, and the suspension obtained is stirred for a few minutes at 20 ^° C. and then cooled to 0 ° C. The milky suspension of activated polymer is then added to this suspension, and the reaction mixture is stirred for 2 h at 0 ^° C. 1.2 ml of ethanolamine is added, and then stirred overnight at 20 ^° C. After adding 2 1 mL of a 35% HCl solution and then 200 mL of water, the reaction mixture is poured dropwise into 700 mL of water, while adjusting the pH to 7.4. The solution obtained is diafiltered against 8 volumes of salt water (0.9%) then 4 volumes of water, and concentrated to a volume of about 250 mL. The percentages of argininamide and ethanolamine grafted, determined by proton NMR in D ₂ O, are 40 and 45%, respectively. f) Synthesis of a cationic PE2-D polyelectrolyte polymer (alpha-tocopherol grafted polyglutamate of synthetic origin, arginine and ethanolamine)

Indices and groups: T = D, L-α-tocopherol, p = 11, q = 48, r = 150, s = 11

Ten grams of a poly (glutamic acid) of DP 220 grafted at 5% randomly with racemic α-tocopherol are solubilized in 125 mL of NMP at 80 ^° C. This solution is cooled to 0 ^° C., and 8.7 ml of tert-butyl chloroformate and 7.3 ml of N-methyl morpholine are added. This reaction mixture is stirred for 15 min at 0 ^° C. In parallel, 4.61 g of argininamide dihydrochloride are suspended in 58 ml of NMP and 2.9 ml of triethylamine are added. Another 2.8 ml of ethanolamine is added, and the suspension obtained is stirred for a few minutes at 20 ^° C. and then cooled to 0 ^° C. The milky suspension of activated polymer is then added to this suspension, and the reaction mixture is stirred for 4 h to 0 ⁰ C. 1.2 ml of ethanolamine are added, and then stirred overnight at 20 ^° C. After addition of 2.1 ml of a 35% solution of HCl, the reaction mixture is poured dropwise. drop in 730 mL of water, while adjusting the pH to 7.4. The solution obtained is diaflltrée against 8 volumes of salt water (0.9%) then 4 volumes of water, and concentrated to a volume of about 300 mL. The percentage of grafted argininamide and ethanolamine, determined by proton NMR in D ₂ O, are 22% and 68%, respectively.

2) Example 1 (comparative): preparation of particles with polyelectrolytes having no hydrophobic group (1) Preparation of a colloidal solution of PEl-B polymer:

The PEl-B polymer obtained according to the synthesis b) above is used. This polymer has a half-neutralization pH equal to 5.985. A colloidal solution of polymer PE1-B is obtained by solubilizing it in water by adjusting the pH to 7.63 by adding a solution of NaOH. The osmolarity of the solution is adjusted to 100 mOsm by introducing the necessary amount of an aqueous solution of NaCl. The polymer concentration PE1-B is adjusted to 8.38 mg / g.

(2) Association of the protein with PEl-B polymer:

To the above colloidal solution of PEl-B polymer is added concentrated IFN-α protein at 2.4 mg / g. The combination having the following characteristics is obtained:

The combination is carried out overnight at 25 ⁰ C with stirring.

(3) Preparation of a colloidal solution of poly-L-arginine (Aldrich P7637):

This polymer has a pH of half-neutralization greater than 9. A colloidal solution of poly-L-arginine is obtained by solubilizing it in water by first adjusting the pH to 0.92 with a solution of HCl, then back at pH equal to 6.91 with a solution of NaOH and heating the solution at 45 ⁰ C for 15 min. The poly-L-arginine polymer concentration is adjusted to 5.13 mg / g.

(4) Mixing to obtain the particles

1.37 g of the poly-L-arginine solution are added dropwise with stirring over 1.06 g of the IFN-α / PEI-B solution at 45 ^° C. 15 min at 45 ^° C. Then, it is stirred overnight at ^40.degree .

Table I below groups together the characteristics of the particles obtained:

The charge ratio Z is the ratio of the number of moles of cationic ionized groups to the number of moles of anionic ionized groups, measured at pHm equal to 6.95. The particle size is measured according to the T test. Table I

(5) Quantification of the encapsulation of the protein

The suspension is centrifuged for 15 min at 8000 rpm and the IFN-α is assayed in the supernatant by the method described in the European Pharmacopoeia (colorimetric assay by UV absorbance).

Almost a third of the introduced protein is not encapsulated in the formed microparticles. This proportion can not lead to a controlled release.

3) Example 2 Preparation of Particles with a Single Polyelectrolyte (PE1) Having Hydrophobic Groups

(1) Preparation of a colloidal solution of PEl-A polymer: The PEl-A polymer obtained according to the synthesis a) above is used. This polymer has a half-neutralization pH equal to 5.445.

A colloidal solution of PEI-A polymer is obtained by solubilizing it in water by adjusting the pH to 7.53 by adding a solution of NaOH. The osmolarity of the solution is adjusted to 101 mOsm by introducing the necessary amount of an aqueous solution of NaCl. The polymer concentration PEI-B is adjusted to 8.41 mg / g.

(2) Association of the protein with PEI-A polymer:

To the colloidal solution of the previous PEI-A polymer, the IFN-α protein concentrated at 2.4 mg / g was added. The combination having the following characteristics is obtained:

The combination is carried out overnight at 25 ° C with stirring.

(3) Preparation of a colloidal solution of poly-L-arginine (Aldrich P7637): The solution is prepared in the same manner as described in Example 1.

(4) Mixing to obtain the particles

1.24 g of the poly-L-arginine solution are added dropwise with stirring over 1.07 g of the IFN-α / PEI-A solution at 45 ^° C. The mixture is stirred for 15 minutes at 45 ° C. . Then, it is stirred overnight at ^40.degree .

Table II below groups together the characteristics of the particles obtained:

The charge ratio Z is measured at pHm equal to 6.88. The particle size is measured according to the T test.

Table II

(5) Quantification of the encapsulation of the protein

The suspension is centrifuged for 15 min at 8000 rpm and PIFN-α is assayed in the supernatant by the method described in the European Pharmacopoeia (colorimetric assay by UV absorbance).

All of the introduced protein is encapsulated in the formed microparticles.

4) Example 3: Preparation of particles based on PEI-A and PE2-A, containing IFN-a 4.1) Example 3.1: final concentration of polymer approximately equal to 10 mg / g, Z being equal to about 1

(1) Preparation of a colloidal solution of polymer PEI-A:

The PEI-A polymer obtained according to the synthesis a) above is used. This polymer has a half-neutralization pH equal to 5.445.

A colloidal solution of PEI-A polymer is obtained by solubilizing it in water by adjusting the pH to 7.45 by adding a solution of NaOH. The osmolarity of the solution is adjusted to 108 mOsm by introducing the necessary amount of an aqueous solution of NaCl. The PEl polymer concentration is adjusted to 23.88 mg / g.

(2) Association of the protein with PEI-A polymer:

To the previous colloidal solution of PEl-A polymer was added concentrated IFN-α protein at 2.4 mg / g and NaCl at 89 mOsm. The combination having the following characteristics is obtained:

The combination is carried out overnight at 25 ⁰ C with stirring. The association is then adjusted to pH equal to 5.07.

(3) Preparation of a colloidal solution of PE2-A polymer:

The polymer PE2-A obtained according to the synthesis c) above is used. This polymer has a half-neutralization pH of 6.05.

A colloidal solution of PE2-A polymer is obtained by solubilizing it in water by adjusting the pH to 5.17. The osmolarity of the solution is adjusted to 289 mOsm and the polymer concentration PE2-A is adjusted to 5.70 mg / g.

(4) Mixing to obtain the particles

4.98 g of the PE2-A solution are added dropwise with stirring to 4.61 g of the IFN-α / PEI-A solution. It is stirred overnight at ^{40 °} C.

Table III below summarizes the characteristics of the particles obtained:

The charge ratio Z is measured at pHm equal to 5.17. The particle size is measured according to the T test.

Table III

4.2) Example 3.2: final polymer concentration equal to 5 mg / g, Z being equal to about 10

(1) Preparation of a colloidal solution of polymer PEI-A:

The PEI-A polymer obtained according to the synthesis a) above is used. A colloidal solution of PEl polymer is obtained by solubilizing it in water by adjusting the pH to 7.52 by adding a solution of NaOH. The osmolarity of the solution is adjusted to 108 mOsm by introducing the necessary amount of an aqueous solution of NaCl. The PEl polymer concentration is adjusted to 20.21 mg / g.

(2) Association of the protein with PEI-A polymer:

To the previous colloidal solution of PEl-A polymer was added concentrated IFN-α protein at 2.4 mg / g and NaCl at 89 mOsm. The combination having the following characteristics is obtained:

The combination is carried out overnight at 25 ⁰ C with stirring. The association is then adjusted to a pH of 4.88.

(3) Preparation of a colloidal solution of polymer PE2-A: The polymer PE2-A obtained according to the synthesis c) above is used. A colloidal solution of PE2-A polymer is obtained by solubilizing it in water by adjusting the pH to 5.07. The osmolarity of the solution is adjusted to 287 mOsm and the polymer concentration PE2-A is adjusted to 8.11 mg / g.

(4) Mixing to obtain the particles: 5.19 g of the PE2-A solution are added dropwise with stirring to 5.02 g of the IFN-α / PEI-A solution. It is stirred overnight at ^{40 °} C.

Table IV below summarizes the characteristics of the particles obtained:

The charge ratio Z is measured at pHm equal to 4.81. The particle size is measured according to the T test.

Table IV

4.3) Example 3.3: final polymer concentration equal to 10 mg / g, Z being equal to about 10

(1) Preparation of a colloidal solution of PEl-A polymer: The PEl-A polymer obtained according to the synthesis a) above is used.

A colloidal solution of PEI-A polymer is obtained by solubilizing it in water by adjusting the pH to 7.52 by adding a solution of NaOH. The osmolarity of the solution is adjusted to 108 mOsm by introducing the necessary amount of an aqueous solution of NaCl. The polymer concentration PEI-A is adjusted to 20.21 mg / g.

(2) Association of the protein with PEI-A polymer:

To the previous colloidal solution of PEl-A polymer was added concentrated IFN-α protein at 2.4 mg / g and NaCl at 89 mOsm. The combination having the following characteristics is obtained:

The combination is carried out overnight at 25 ⁰ C with stirring. The association is then adjusted to pH equal to 5.07.

(3) Preparation of a colloidal solution of polymer PE2-A: The polymer PE2-A obtained according to the synthesis c) above is used. A colloidal solution of PE2-A polymer is obtained by solubilizing it in water by adjusting the pH to 5.08. The osmolarity of the solution is adjusted to 288 mOsm and the polymer concentration PE2-A is adjusted to 16.37 mg / g.

(4) Mixing to obtain the particles:

5.25 g of the PE2-A solution are added dropwise with stirring to 5.19 g of the IFN-α / PEI-A solution. It is stirred overnight at ^{40 °} C.

Table V below summarizes the characteristics of the particles obtained:

The charge ratio Z is measured at pHm equal to 4.95. The particle size is measured according to the T test.

Table V

S) Example 4 Preparation of Particles Based on PEI-A and PE2-B, Containing

IFN-α, final polymer concentration equal to 5 mg / g, Z = I

(1) Preparation of a colloidal solution of PEl-A polymer: The PEl-A polymer obtained according to the synthesis a) above is used.

A colloidal solution of PEI-A polymer is obtained by solubilizing it in water by adjusting the pH to 7.52 by adding a solution of NaOH. The osmolarity of the solution is adjusted to 108 mOsm by introducing the necessary amount of an aqueous solution of NaCl. The polymer concentration PEI-A is adjusted to 20.21 mg / g.

(2) Association of the protein with PEI-A polymer:

To the previous colloidal solution of PEl-A polymer was added concentrated IFN-α protein at 2.4 mg / g and NaCl at 89 mOsm. The combination having the following characteristics is obtained:

The combination is carried out overnight at 25 ⁰ C with stirring. The association is then adjusted to pH equal to 6.89.

(3) Preparation of a colloidal solution of PE2-B polymer:

The PE2-B polymer obtained according to the synthesis d) above is used. This polymer has a neutralization pH greater than 9.

A colloidal solution of PE2-B polymer is obtained by solubilizing it in water by adjusting the pH to 6.98. The osmolarity of the solution is adjusted to 288 mOsm and the PE2-B polymer concentration is adjusted to 6.33 mg / g.

(4) Mixing to obtain the particles:

4.06 g of the PE2-B solution are added dropwise with stirring to 4.59 g of the IFN-α / PEI-A solution. It is stirred overnight at ^{40 °} C.

Table VI below groups together the characteristics of the particles obtained:

The charge ratio Z is measured at pHm equal to 6.85. The particle size is measured according to the T test.

Table VI

6) Example 5 (comparative): preparation of particles based on PEI-A alone, containing VIFN a

The PEI-A polymer obtained according to the synthesis a) above is used. A colloidal solution of PEI-A polymer is obtained by solubilizing it in water by adjusting the pH to 7.52 by adding a solution of NaOH. The osmolarity of the solution is adjusted to 108 mOsm by introducing the necessary amount of an aqueous solution of NaCl. The polymer concentration PEI-A is adjusted to 29.05 mg / g.

To the above colloidal solution of PEl polymer was added IFN-α protein concentrated at 2.4 mg / g. The combination is carried out overnight at 25 ⁰ C with stirring. Table VII below groups together the characteristics of the particles obtained: The particle size is measured according to the T 'test.

Table VII

7) In Vitro Results for Examples 2, 3, 4 and 5

For this purpose, the release of the active ingredient from the particles according to the invention is measured using the L-test.

Figure 1 shows the release in the L-test as the percentage of protein released over time.

The formulation of Comparative Example 2 in which only one of the polymers carrying hydrophobic groups has a very low release profile, with 1.6% of the protein released after 23 hours.

The formulation of Comparative Example 5, containing 23 mg / g of PEl particles has a profile similar to the particles of Example 3.1 (PE1 / PE2-A, Z = I at 10 mg / g)

(respectively 93% in 10 hours and 72% in 48 hours).

In the case of the particles of Examples 3.2 (PE1 / PE2-A, Z = 10 and 5 mg / g) and 3.3

(PE1 / PE2-A, Z = 10 and 10 mg / g), we observe the creation of a constant release flux, which is not zero at the end of the experiment: respectively 65 and 19% of the protein injected in 48 hours.

In the case of the particles of Example 4 (PE1 / PE2-B, Z = 1 and 5 mg / g), the creation of a constant flow of release is observed, which is not zero at the end of the experiment, with a release of 7% of the injected protein in 48 hours.

8) In Vivo Results for Examples 3, 4 and 5

44 rats were separated into 5 groups of 8 or 12 animals and received in parallel an IR immediate release formulation or sustained release formulation corresponding to Comparative Example 5 or one of the formulations of Examples of Invention 3 and 4. at a dose of 300 μg / kg. The pharmacokinetic results are summarized in Table VIII below:

Table VIII

C _max represents the maximum average plasma concentration of protein on all animals.

Median T _max represents the median time for which the plasma concentration passes through its maximum. AUC represents the mean area under the curve of plasma concentration as a function of time.

T50% _AUC represents the average time at which the area under the curve reaches 50% of its total value.

RBA is the ratio of the area under the curve of the formulation considered to the area under the curve of the IFN IR formulation.

All the formulations have a prolonged release profile accompanied by a decrease in C _max compared to IR.

With the exception of the formulation of Example 3.1 (PE1 / PE2-A, Z = I at 10 mg / g) whose release profile is close to that of the formulation of Comparative Example 5, the terminal slope is lower, indicating prolonged residual absorption.

It will be noted for the formulation of Example 3.3 (PE1 / PE2-A, Z = 10 and 10 mg / g) a release up to more than one week.

9) EXAMPLE 6 Preparation of PEl-A and PE2-C-based Particles Containing IFN-α, Final Polymer Concentration of 5 mg / g, Z = I

(1) Preparation of a colloidal solution of PEI-A polymer The PEI-A polymer obtained according to the synthesis a) above is used. A colloidal solution of polymer PEI-A is obtained by solubilizing it in water by adjusting the pH to 7.15 by adding a solution of NaOH. The osmolarity of the solution is adjusted to 145 mOsm by introducing the necessary amount of an aqueous solution of NaCl. The polymer concentration PEI-A is adjusted to 3.10 mg / g.

(2) Combination of Therapeutic Protein with PEl-A Polymer: The above colloidal polymer PEl-A solution of concentrated IFN-α protein was added to 2.7 mg / g. The combination having the following characteristics is obtained:

The combination is carried out overnight at 25 ° C with stirring. The association is then adjusted to a pH of 7.0.

(3) Preparation of a colloidal solution of PE2-C polymer:

The PE2-C polymer obtained according to the synthesis e) above is used. A colloidal solution of PE2-C polymer is obtained by diluting and adjusting the PE2-C polymer to pH 7.04, 288 mOsm and 7.96 mg / g in 140 mOsm PBS.

(4) Mixing to obtain the particles:

15.14 g of the PE2-C solution are added dropwise with stirring to 16.374 g of the IFN-α / PEI-A solution. It is stirred overnight at ^{40 °} C.

The charge ratio Z is measured at pHm equal to 7. The size of the particles is measured according to the T test. The table below groups together the characteristics of the particles obtained:

10) Example 7 Preparation of PEI-A and PE2-D-Based Particles Containing IFN-α, Final Polymer Concentration of 5 mg / g, Z = 1

(1) Preparation of a colloidal solution of PEl-A polymer: The PEl-A polymer obtained according to the synthesis a) above is used. A colloidal solution of polymer PEI-A is obtained by solubilizing it in water by adjusting the pH to 7.02 by addition of a solution of NaOH. The osmolarity of the solution is adjusted to 101 mOsm by introducing the necessary amount of an aqueous solution of NaCl. The polymer concentration PEI-A is adjusted to 2.0 mg / g.

(2) Association of the protein with PEI-A polymer:

To the above colloidal solution of PEl-A polymer, the IFN-α protein concentrated at 2.7 mg / g was added. The combination having the following characteristics is obtained:

The combination is carried out overnight at 25 ⁰ C with stirring. The association is then adjusted to a pH of 7.0.

(3) Preparation of a colloidal solution of polymer PE2-D: The polymer PE2-D obtained according to the synthesis f) above is used. A colloidal solution of PE2-D polymer is obtained by diluting the PE2-D polymer in water and adjusting to pH 7 with 0.1 N HCl or 0.1 N NaOH. The concentration of PE2-D polymer is adjusted. , 82 mg / g.

(4) Mixing to obtain the particles:

1.2 g of the PE2-D solution are added dropwise with stirring to 1.2 g of the IFN-α / PEI-A solution. It is stirred overnight at ^{40 °} C.

The charge ratio Z is measured at pHm equal to 7. The size of the particles is measured according to the T test. The table below groups together the characteristics of the particles obtained:

11) Example 8: (Comparison) release rate of the PE1-A / PE2-D-based and PE1-A / PE2-C-containing particles containing IFN-α.

The particles obtained by the preparations described in Examples 6 and 7 above are compared in the continuous flow cell release test L described above. The table below groups the results obtained:

In conclusion, this example shows that it is possible, in particular by selecting a cationic polymer containing more or fewer neutral and / or anionic groups, to modulate the release rate of the AP. Thus, at 12 h, the release is 9.5% for the microparticles obtained from the PE2-C polymer and 31% for the microparticles obtained from PE2-D.

Claims

Particles for the sustained release of at least one active principle (AP), characterized in that they comprise: a) a first polyelectrolyte polymer (PEI), preferably a linear alpha-polyamino acid, in the charged state, carrying lateral hydrophobic groups (GH), said first polyelectrolyte polymer (PE1) spontaneously forming in water a colloidal solution of particles at at least a pHm value of pH of between 3 and 8; b) a second polyelectrolyte polymer (PE2), preferably a linear alpha-polyamino acid, of opposite polarity to that of the first polyelectrolyte polymer (PE1), said second polyelectrolyte polymer (PE2) forming a solution or a colloidal solution in water; minus said pHm value of the pH; with the proviso that, if the first electrolyte polymer (PE1) is a polyamino acid, then the second polyelectrolyte polymer (PE2) is neither polylysine nor polyethylene imine; c) at least one active ingredient (PA) non-covalently associated with the particles of the colloidal solution of the first polyelectrolyte polymer (PEI); said particles for the sustained release of at least one active ingredient (PA) being obtained by mixing, at pH equal to pHm, the first polyelectrolyte polymer (PEI) in the form of a colloidal solution of particles associated with the active principle (PA) with the second polyelectrolyte polymer (PE2) in the form of a solution or colloidal solution.

2. Particles for the sustained release of at least one active principle (AP), characterized in that they comprise: a) a first polyelectrolyte polymer (PEI), preferably a linear alpha-polyamino acid, in the charged state, carrying lateral hydrophobic groups (GH), said first polyelectrolyte polymer (PE1) spontaneously forming in water a colloidal solution of particles at at least a pHm value of pH of between 3 and 8; b) a second polyelectrolyte polymer (PE2), preferably a linear alpha-polyamino acid, of opposite polarity to that of the first polyelectrolyte polymer (PE1), carrying hydrophobic groups (GH) on the side, said second polyelectrolyte polymer (PE2) forming in the water a solution or a colloidal solution at at least said pHm value of the pH; c) at least one active ingredient (PA) non-covalently associated with the particles of the colloidal solution of the first polyelectrolyte polymer (PEI); said particles for the sustained release of at least one active ingredient (PA) being obtained by mixing, at pH equal to pHm, the first polyelectrolyte polymer (PEI) in the form of a colloidal solution of particles associated with the active principle (PA) with the second polyelectrolyte polymer (PE2) in the form of a solution or colloidal solution.

3. Particles according to claim 1 or 2, characterized in that the first and second polyelectrolyte polymers (PE1, PE2) are polyamino acids, or a pharmaceutically acceptable salt thereof, the main chain of which is formed by residues chosen from the group of aspartic residues, glutamic residues and their combinations, at least a portion of these residues being modified by grafting at least one hydrophobic group (GH) for at least the first polyelectrolyte polymer (PEI).

4. Particles according to claim 3, characterized in that one of the polyelectrolyte polymers (PE1, PE2), or one of their pharmaceutically acceptable salts, corresponds to the following formula (I):

[BOY WUT

(I) in which:

R ¹ represents H, linear C ₂ -C ₁₀ or branched C ₃ -C ₁₀ alkyl, benzyl, -R ⁴ - [GH], or R ¹ forms with NH a terminal amino acid residue; R ² represents H, linear C ₂ to C ₁₀ or branched C ₃ to C ₁₀ acyl, pyroglutamate or -R ⁴ - [GH];

R ⁴ represents a direct bond or a "spacer" based on 1 to 4 amino acid residues;

A and A independently represent -CH ₂ - or -CH ₂ -CH ₂ -; n / (n + m) is defined as the molar grafting level and its value is sufficiently low for the polymer dissolved in water at pH 7 and at 25 ^° C. to form a colloidal suspension of polymer particles; n + m varies from 10 to 1000, preferably from 50 to 300; GH represents a hydrophobic group having 6 to 30 carbon atoms or is selected from the following group of radicals: (I) alkyls, acyls or alkenyls linear or branched, preferably linear C ₁ -C ₂₀ and, more preferably still C ₂ -C 18;

(ii) hydrocarbon groups containing one or more heteroatoms, preferably those containing oxygen and / or sulfur and, more preferably, those of the following formula:

H. H

- ^ C - CHI-I ^ CCH ₂₀ * - C- CH ₂ OR ₁

R 60 R, 61

in which:

_6O R is an alkyl, acyl or linear or branched alkenyl, preferably linear C ₁ -C ₂₀ and even more preferably C ₂ -C _S,

R _6I and R ₆₂ are identical to or different from each other and correspond to hydrogen or to a linear or branched, preferably linear C ₁ -C ₂₀ and even more preferably C ₂ -C ₈ alkyl, acyl or alkenyl radical, q = 1 to 100;

(iii) aryls, aralkyls or alkylaryls, preferably aryls;

(iv) the hydrophobic derivatives, preferably the phosphatidylethanolamino radical or the radicals chosen from octyloxy-, dodecyloxy-, tetradecyloxy-, hexadecyloxy-, octadecyloxy-, 9-octadecenyloxy-, tocopheryloxy- or cholesteryloxy-.

5. Particles according to claim 3, characterized in that the polyelectrolyte polymer PE2, or a pharmaceutically acceptable salt thereof, corresponds to one of the following formulas (II), (III) and (IV):

in which :

GH represents a hydrophobic group comprising 6 to 30 carbon atoms;

R ³⁰ is a linear C ₂ -C ₆ alkyl;

R ⁵⁰ is alkyl, dialkoxy or C ₂ -C ₆ diamino;

• R ⁴ represents a direct bond or a "spacer" based on 1 to 4 amino acid residues;

"A ¹ and A ² independently represent -CH ₂ - or -CH ₂ -CH ₂ -;

• n '+ m' or n "is defined as the degree of polymerization and varies from 10 to 1000, preferably between 50 and 300.

6. Particles according to claim 3, characterized in that one of the polyelectrolyte polymers (PE1, PE2), or a pharmaceutically acceptable salt thereof, corresponds to the following formula (V):

in which :

E- is independently: -NHR wherein R is H, linear C ₂ -C ₁₀ or branched C ₃ -C ₁₀ alkyl or benzyl, amino acid residue or terminal amino acid derivative of formula :

H -NH - C COR ⁷

wherein R ⁷ is OH, OR ⁹ or NHR ¹⁰ , and R ⁸ , R ⁹ and R ¹⁰ are independently H, linear C ₂ -C ₁₀ or branched C ₃ -C 10 alkyl or benzyl;

B is a direct bond or a divalent, trivalent or tetravalent linking group, preferably chosen from the following radicals:

-O-, -NH-, -N (C _1-5 alkyl) -, an amino acid (preferably natural), diol, triol, diamine, triamine, aminoalcohol or hydroxy acid residue comprising from 1 to 6 carbon atoms;

D is H, linear C ₂ -C 10 acyl, branched C ₃ -C 10 acyl, or pyroglutamate;

GH represents a hydrophobic group having 6 to 30 carbon atoms;

R ⁷⁰ represents a radical chosen from the following group:

-NH- (CH ₂ ) _W -NH ₃ ⁺ with w between 2 and 6, and preferably w is equal to 4,

- -NH- (CH ₂ ) ₄ -NH-C (= NH) -NH ₃ ⁺ ,

- -O- (CH ₂ ) ₂ -NH ₃ ⁺ ,

-O- (CH ₂ ) ₂ -N ⁺ (CH ₃ ) ₃ ,

or

an alkyl ester (preferably -COOMe and -COOEt), -CH ₂ OH, -C (≈O) -NH ₂ , -C (= O) -NH-CH ₃ or -C (= O) -N (CH ₃ ) ₂ ; an amino acid residue or amino acid derivative of formula:

in which :

X is an oxygen atom or -NH-, R ¹² is H, linear C ₂ -C ₁₀ alkyl, branched C ₃ -C ₁₀ alkyl or benzyl,

R ¹³ is - (CH ₂ ) ₄ NH ₃ ⁺ , - (CH ₂ ) ₃ NH-C (= NH) -NH ₃ ⁺ , - (CH ₂ ) ₃ NH ₃ ⁺ ; the counteranion of R is a chloride, a sulfate, a phosphate or an acetate, preferably a chloride; R ⁹⁰ represents a hydroxyethylamino, an alkylene glycol residue or a polyoxyalkylene;

• p, q, r and s are positive integers;

(p) / (p ^{+ c} 1 ^{+ r + s} ) ^is defined as the molar grafting ratio of the hydrophobic groups GH varies from 2 to 99 mol%, and preferably between 5 and 50%, provided that each chain of The copolymer has on average at least 3 hydrophobic grafts;

• (q) / (p + q + r + s) is defined as the molar grafting rate of the cationic groups and varies from 1 to 99 mol%;

"(p + q + r + s) ranges from 10 to 1000, preferably from 30 to 500;" (r) / (p + q + r + s) ranges from 0 to 98 mol%;

• (s) / (p + q + r + s) varies from 0 to 98 mol%.

7. Particles according to any one of claims 4 to 6, characterized in that R or B represent a direct bond.

8. Particles according to any one of the preceding claims, characterized in that all or part of the hydrophobic groups (GH) are independently selected from the following group of radicals:

"a linear or branched alkoxy having from 6 to 30 carbon atoms and which may comprise at least one heteroatom (preferably O and / or N and / or S) and / or at least one unsaturation,

An alkoxy having 6 to 30 carbon atoms and having one or more annealed carbocycles and optionally containing at least one unsaturation and / or at least one heteroatom (preferably O and / or N and / or S), An alkoxyaryl or an aryloxyalkyl of 7 to 30 carbon atoms and which may comprise at least one unsaturation and / or at least one hetero atom (preferably O and / or N and / or S).

9. Particles according to any one of claims 4 to 8, characterized in that the hydrophobic groups (GH) each independently represent a monovalent radical of the following formula:

(GH) in which:

- R ⁵ represents a methyl (alanine), isopropyl (valine), isobutyl (leucine), secbutyl (isoleucine), benzyl (phenylalanine);

- R ⁶ represents a hydrophobic radical containing from 6 to 30 carbon atoms; - 1 varies from 0 to 6.

10. Particles according to claim 9, characterized in that all or part of the hydrophobic radicals R ⁶ of the hydrophobic groups (GH) are independently selected from the following group of radicals: "a linear or branched alkoxy having from 6 to 30 atoms of carbon and which may comprise at least one heteroatom (preferably O and / or N and / or S) and / or at least one unsaturation,

an alkoxy having 6 to 30 carbon atoms and having one or more annealed carbocycles and optionally containing at least one unsaturation and / or at least one heteroatom (preferably O and / or N and / or S),

an alkoxyaryl or an aryloxyalkyl of 7 to 30 carbon atoms and may comprise at least one unsaturation and / or at least one heteroatom (preferably O and / or N and / or S).

11. Particles according to any one of the preceding claims, characterized in that the polymer PE1 or PE2 comprises simultaneously: - from 15 to 50 mol% of glutamate monomers; from 20 to 55 mol% of nonionizable monomers such as groups substituted with a hydroxyethylamino radical; from 10 to 40 mol% of monomers carrying cationic groups of neutralization pH greater than 8; from 3 to 15 mol% of nonionizable monomers substituted with a hydrophobic group.

12. Particles according to claim 11, characterized in that the polymer PE1 or PE2 is cationic and comprises simultaneously: from 0 to 5 mol% of glutamate monomers; from 50 to 85 mol% of nonionizable monomers such as groups substituted with a hydroxyethylamino radical; from 10 to 40 mol% of monomers carrying cationic groups of neutralization pH greater than 8; from 3 to 15 mol% of nonionizable monomers substituted with a hydrophobic group.

13. Particles according to any one of the preceding claims, characterized in that they have, at physiological pH, a size, measured in a T test, between 1 and 100 microns.

14. Particles according to any one of the preceding claims, characterized in that they have, at physiological pH, a bulk density of between 0.15 and 1.1.

15. Process for the preparation of particles for the sustained release of at least one active principle (AP), these particles being in particular according to any one of Claims 1 and 3 to 14, comprising the following steps:

1) the preparation, at a pHm value of pH between 3 and 8, of an aqueous colloidal solution of a first polyelectrolyte polymer (PE1) in the charged state, carrying hydrophobic groups (GH) on the side, said first polymer polyelectrolyte (PE1) being capable of spontaneously forming in water a colloidal solution of particles with at least a pH value, pHm, of pH between 3 and 8;

2) the addition of at least one active ingredient (AP) to the first polyelectrolyte polymer (PEI) obtained in step 1, said active ingredient associating non-cumulatively with the particles of the colloidal solution of said first polymer polyelectrolyte (PEI);

3) preparing a second polyelectrolyte polymer (PE2) of opposite polarity to that of the first polyelectrolyte polymer (PEI), said second polyelectrolyte polymer (PE2) forming in water a solution or colloidal solution at at least said pHm value of the pH, provided that if the first electrolyte polymer (PE1) is a polyamino acid, then the second polyelectrolyte polymer (PE2) is neither polylysine nor polyethylene imine;

4) mixing, at pH equal to pHm, the first polyelectrolyte polymer (PEI) in the form of a colloidal solution of particles to which the active ingredient (PA) obtained in step 2) is combined with the second polyelectrolyte polymer (PE2) under solution form or colloidal solution obtained in step 3).

16. A process for preparing particles for the sustained release of at least one active principle (AP), said particles being in particular according to any one of claims 2 to 12, comprising the following steps: 1) the preparation, at a a pHm value of the pH between 3 and 8, of an aqueous colloidal solution of a first polyelectrolyte polymer (PE1) in the charged state, bearing hydrophobic groups (GH) on the side, said first polyelectrolyte polymer (PE1) being capable of spontaneously forming in water a colloidal solution of particles with at least a pH value of pH of between 3 and 8;

2) adding at least one active ingredient (AP) to the first polyelectrolyte polymer (PEI) obtained in step 1, said active ingredient non-covalently associating with the particles of the colloidal solution of said first polyelectrolyte polymer ( PEI); 3) preparing a second polyelectrolyte polymer (PE2) of opposite polarity to that of the first polyelectrolyte polymer (PE1), carrying hydrophobic groups (GH) on the side, said second polyelectrolyte polymer (PE2) forming in water a solution or a colloidal solution at at least said pHm value of the pH; 4) mixing, at pH equal to pHm, the first polyelectrolyte polymer (PEI) in the form of a colloidal solution of particles to which the active ingredient (PA) obtained in step 2) is combined with the second polyelectrolyte polymer (PE2) under solution form or colloidal solution obtained in step 3).

17. Pharmaceutical formulation for the sustained release of at least one active principle (AP), characterized in that it comprises an aqueous suspension of particles according to any one of claims 1 to 14 or those obtained by the method according to any one of claims 15 to 16.

18. Solid pharmaceutical formulation for the sustained release of at least one active principle (AP), characterized in that it comprises a dry powder form:

• based on particles containing at least one active ingredient (AP), these particles being those according to any one of claims 1 to 14 or those obtained by the method according to any one of claims 15 to 16;

Or obtained from the formulation according to claim 17.

19. A process for preparing medicaments, in particular for parenteral, mucosal, subcutaneous, intramuscular, intradermal, transdermal intraperitoneal, intracerebral or tumor administration, or even orally, nasally, pulmonary, vaginally or ocularly, characterized in that it essentially consists in implementing at least one formulation according to any one of claims 17 or 18.